Smooth muscle is finely tuned to carry out mechanical functions specific to the different hollow organs and vasculature they surround. Altering the contractile behaviors of smooth muscle cells can lead to a variety of pathophysiological states, such as hypertension resulting in cardiac failure and airway hyperresponsiveness associated with asthma. Mechanisms implicated in these disease states include smooth muscle plasticity, myosin isoform shifts, and impaired regulation. In each case, the proximal cause is hypercontractility generated by a change in actin-myosin activity. Actin-myosin activity is primarily regulated through the phosphorylation of smooth muscle myosin (SMM). Specifically, phosphorylation of SMM by myosin light chain kinase (MYLK) activates actin-myosin ATPase activity and muscle contraction. In this way, the activity of SMM is directly linked to the activity of MYLK;in fact MYLK activity appears to exert tight control over smooth muscle activity. It is thus no surprise that small changes in MYLK activity have been directly linked to many chronic and acute human diseases. Yet remarkably little is known about the factors that influence MYLK activity. Our preliminary studies indicate that MYLK-SMM interactions limit MYLK activity. In this proposal we continue to test this hypothesis and determine the factors that influence MYLK- SMM interactions. We will use an in vitro model system to control the constituents of our system and use a wide range of biochemical, kinetic, and imaging techniques to simultaneously measure MLYK-SMM interactions, SMM phosphorylation, and activation of mechanics. We will use solution kinetics to establish more detailed kinetic mechanisms and cell studies to establish in vivo relevance. This proposal will provide direct measurements of the mechanisms by which MYLK tunes smooth muscle contraction in normal and disease states. Moreover, the insights developed through this proposal will extend of knowledge of how non-muscle myosin is activated in non-muscle cells. PUBLIC HEALTH RELEVANCE: Changes in smooth muscle mechanics are associated with a variety of diseases, such as hypertension and asthma. Myosin light chain kinase (MYLK) is an enzyme that activates smooth muscle mechanics by phosphorylating smooth muscle myosin - the proteins that generate force in muscle. In this proposal, we will investigate key factors that influence MYLK activity as a first step in developing new therapies for regulating smooth muscle mechanics.